During the past 9 months we have completed patient entry into the low-dose cyclophosphamide (Cy) trial. The following parameters of the immune response were followed to gauge the effect of cytoxan doses: skin tests for delayed hypersensitivity to recall antigens (Mumps, PPD, Candida, SK/SD, and DNCB); antibody titers produced in vivo as a consequence of vaccination with Fluogen or Pnu-Immune (pneumococcal) vaccines; determination of T-cell subsets; mixed lymphocyte culture (MLC) response; and antibody production against SRBC by peripheral blood lymphocytes as measured by a plaque-forming cell assay. Skin tests for delayed hypersensitivity to recall antigens, T-cell subsets, and MLC response gave no indication of an increase in suppressor cell number of activity prior to treatment and no change was seen after treatment. No increase in antibody response against flu or pneumococcal antigens was seen. Testing of pre-\and post-treatment sera for antibody titers against melanoma antigens is in progress. The plaque-forming cell assay results, however, were markedly affected by Cy treatment. Of 14 patients who showed suppressor cell activity initially, nine showed normalization of the PFC response, 4/4 receiving 500mg/M2 of Cy, 4/7 receiving 300mg/M2, and 1/3 receiving 100mg/M2. The maximal effect was seen 2 to 4 weeks after treatment. Median numbers of PFC seen in responders before and after treatment were as follows: 500mg/M2 -\0,80; 300mg/M2 -\0,96; 100mg/M2 -\0,95. Unexpectedly, an even greater effect was observed when PBL were passed over Sephadex G-10 before the assay. Under these circumstances, median PFC numbers were: 500mg/M2 -\51,307; 300mg/M2 -\135,1900; 100mg/M2 -\0,72. Cytoxan had no effect on antibody production in patients who showed a normal response before treatment. These observations show that low doses of Cy have profound effects on immune regulation and that in vitro antibody production against sheep red blood cells provides a sensitive assay for detecting and dissecting these effects. (IT)